2017

Résumé: Variable and low egg quality is a major limiting factor for the development of efficient aquaculture production. This stems from limited knowledge on the mechanisms underlying egg quality in cultured fish. Molecular analyses, such as transcriptomic studies, are valuable tools to identify the most important processes modulating egg quality. However, very few studies have been devoted to this aspect so far. Within this study, the microarray-based transcriptomic analysis of eggs (of different quality) of sea bass (Dicentrarchus labrax) was performed. An Agilent oligo microarray experiment was performed on labelled mRNA extracted from 16 batches of eggs (each batch obtained from a different female) of sea bass, in which over 24,000 published probe arrays were used. We identified 39 differentially expressed genes exhibiting a differential expression between the groups of low (fertilization rate < 60 %) and high (fertilization rate > 60 %) quality. The mRNA levels of eight genes were further analyzed by quantitative PCR. Seven genes were confirmed by qPCR to be differentially expressed in eggs of low and high quality. This study confirmed the importance of some of the genes already reported to be potential molecular quality indicators (mainly rnf213 and irf7), but we also found new genes (mainly usp5, mem-prot, plec, cenpf), which had not yet been reported to be quality-dependent in fish. These results suggest the importance of genes involved in several important processes, such as protein ubiquitination, translation, DNA repair, and cell structure and architecture; these probably being the mechanisms that contribute to egg developmental competence in sea bass.

Résumé: The greater amberjack, Seriola dumerili (Risso, 1810), is a promising candidate for the diversification of European aquaculture production, but inconsistent reproduction in captivity prevents commercial production. Recent studies showed that greater amberjack confined in sea cages exhibited scarce gonad development and early interruption of gametogenic activity during the reproductive season. The aim of the present study was to improve our understanding of the observed impairment of spermatogenesis. Adult wild and captive-reared males were sampled during 3 different phases of the reproductive cycle: early gametogenesis (EARLY; late April to early May), advanced gametogenesis (ADVANCED; late May to early June), and spawning (SPAWNING; late June to July). Spermatogonial stem cells and proliferating germ cells were identified through the immunohistochemical localization of Pou5f1 and proliferating cell nuclear antigen, respectively. Apoptotic germ cells were identified throughout the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling method. Sperm quality of captive-reared fish was evaluated using computer-assisted sperm analysis. Captive-reared males exhibited seminiferous lobules of a smaller diameter, a precocious and progressive decrease of spermatogonial mitosis, and a high level of apoptosis at the beginning of the reproductive season, concomitant with a many-fold higher 17 beta-estradiol plasma concentration. The motile spermatozoa percentage of captive greater amberjack was lower than in other teleosts, and a drastic decrease of spermatozoa motility duration, velocity, and ATP content occurred along the reproductive season. An abnormal increase of sperm concentration as well as an increase of dead spermatozoa occurred during the SPAWNING phase, probably because of lack of sperm hydration and ejaculation and consequent sperm ageing. The present study demonstrates the extreme susceptibility of greater amberjack to rearing stress and underscores the need for improvement of the rearing and handling procedures to ameliorate gametogenesis dysfunctions in commercial aquaculture production.

2016

Résumé: Limited resources in the environment prevent individuals from simultaneouslymaximizing all life-history traits, resulting in trade-offs. In particular, the cost of reproduction is well known to negatively affect energy investment in growth and maintenance. Here, we investigated these trade-offs during contrasting periods of high versus low fish size and body condition (before/after 2008) in the Gulf of Lions. Female reproductive allocation and performance in anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) were examined based onmorphometric historical data from the 1970s and from 2003 to 2015. Additionally, potential maternal effects on egg quantity and quality were examined in 2014/2015. After 2008, the gonadosomatic index increased for sardine and remained steady for anchovy, while a strong decline in mean length at first maturity indicated earlier maturation for both species. Regarding maternal effects, for both species egg quantity was positively linked to fish size but not to fish lipid reserves, while the egg quality was positively related to lipid reserves. Atresia prevalence and intensity were rather low regardless of fish condition and size. Finally, estimations of total annual numbers of eggs spawned indicated a sharp decrease for sardine since 2008 but a slight increase for anchovy during the last 5 years. This study revealed a biased allocation towards reproduction in small pelagic fish when confronted with a really low body condition. This highlights that fish can maintain high reproductive investment potentially at the cost of other traits which might explain the present disappearance of old and large individuals in the Gulf of Lions.

Résumé: Most publications devoted to the cryopreservation of mollusc sperm have focused on the definition of technical protocols, avoiding the description of sperm quality after thawing. The present study investigated the effects of cryopreservation on sperm quality in the great scallop. Wild scallop were fished during the natural spawning period and conditioned in the hatchery before use. Sperm samples were obtained after intragonadal injection of serotonin and cryopreserved using a previously published protocol. Sperm quality was assessed using a panel of four parameters: sperm motility characteristics, using a computer assisted sperm analysis plugin with Image J, intracellular ATP content using an ATP-Lite kit, sperm integrity, using flow cytometry and sperm morphology, using transmission electron microscopy. For each parameter, fresh (control) and thawed spermatozoa were compared. A significant decrease of both the percentage of motile spermatozoa (reduction: 75%) and sperm swimming speed (86%) were observed for thawed sperm compared with fresh sperm. The percentage of living spermatozoa, as assessed using flow cytometry, was significantly lower for thawed sperm (72.4 +/- 2.5%) compared with fresh sperm (86.4 +/- 1.1). However, no significant difference of intracellular sperm ATP content was observed between fresh and thawed sperm. Post thawing, while some spermatozoa showed little or no morphological differences compared with fresh sperm, others had undergone drastic changes, including swelling of the plasma membrane, structural alterations of the chromatin and damage to mitochondria. In conclusion, the descriptive parameters studied in the present work showed that the quality of thawed great scallop sperm was lower than that of fresh cells but was still sufficient for use in aquaculture programs and sperm cryobanking for this species. (c) 2016 Elsevier Inc. All rights reserved.

Résumé: Seasonal changes of sperm quality which can affect sperm biological parameters throughout the breeding period, have been little studied in mollusc species. Controlling gamete quality would aid the management of gametes in hatcheries and the development of selection programs. The aim of the present study was to describe the changes in sperm quality of wild Pacific oysters through the spawning season by comparing sperm parameters al the beginning (May), middle (July) and end (October) of this period using a panel of bio-descriptors. These parameters were studied over the 2014 breeding season based on shed sperm collected after serotonin injection of wild breeders. A significantly higher percentage of motile sperm was observed al the end of the spawning season (+78% relative to the value observed al the beginning) althought a lower total number of spermatozoa was collected (-59%). The mean condition index of oysters, however, was no different between the three sampling dates. For intratesticular sperm, the increase of the percentage of motile sperm and Velocity of the Average Path (VAP) in relation to time post activation was not significantly different among sperm sampling periods, suggesting that the kinetic of the sperm 'maturation process was similar. Furthermore, the mean VAP observed on shed sperm did not change through the spawning season. The sub-continuous gametogenesis of Pacific oyster can help to explain why only limited consequences of sperm ageing are observed in this species. Furthermore, the effects of sperm ageing may depend on the annual reproductive pattern of Pacific oyster. Statement of relevance: This study showed the effect of sperm ageing on sperm quality parameters. This knowledge is useful for aquaculture and would support the recent trends of mollusc farming, allowing a better management of the gametes in hatchery. (C) 2016 Elsevier B.V. All rights reserved.

2015

Résumé: Retinitis pigmentosa 2 (RP2) gene is responsible for up to 20% of X-linked retinitis pigmentosa, a severe heterogeneous genetic disorder resulting in progressive retinal degeneration in humans. In vertebrates, several bodies of evidence have clearly established the role of Rp2 protein in cilia genesis and/or function. Unexpectedly, some observations in zebrafish have suggested the oocyte-predominant expression of the rp2 gene, a typical feature of maternal-effect genes. In the present study, we investigate the maternal inheritance of rp2 gene products in zebrafish eggs in order to address whether rp2 could be a novel maternal-effect gene required for normal development. Although both rp2 mRNA and corresponding protein are expressed during oogenesis, rp2 mRNA is maternally inherited, in contrast to Rp2 protein. A knockdown of the protein transcribed from both rp2 maternal and zygotic mRNA results in delayed epiboly and severe developmental defects, including eye malformations, that were not observed when only the protein from zygotic origin was knocked down. Moreover, the knockdown of maternal and zygotic Rp2 revealed a high incidence of left-right asymmetry establishment defects compared to only zygotic knockdown. Here we show that rp2 is a novel maternal-effect gene exclusively expressed in oocytes within the zebrafish ovary and demonstrate that maternal rp2 mRNA is essential for successful embryonic development and thus contributes to egg developmental competence. Our observations also reveal that Rp2 protein translated from maternal mRNA is important to allow normal heart loop formation, thus providing evidence of a direct maternal contribution to left-right asymmetry establishment.

Résumé: Studying gamete biology can provide important information about a species fertilization strategy as well as their reproductive ecology. Currently, there is a lack of knowledge about how long sea bass Dicentrarchus labrax eggs can remain viable after being activated in seawater. The objectives of this study were to understand the effects of pre-incubation of fresh and overripe sea bass eggs in seawater and to determine the duration of egg receptivity. Pooled eggs (fresh and overripe) from four females were pre-incubated in seawater for 0 min (control), 0.5 min, 1 min, 3 min, 10 min and 30 min and then fertilized by pooled sperm from four males. The fresh eggs had a higher fertilization success than overripe eggs. Our results revealed a significant effect of pre-incubation time for both the fresh (P < 0.01) and overripe eggs (P < 0.01). Fertilization success of eggs significantly declined for both these treatments after 3 min of pre-incubation, which clearly indicates that sea bass eggs are able to be fertilized by sperm for up to 3 min after release into seawater. This study has particular importance for understanding fertilization strategies, reproductive potential, as well as reproductive ecology of sea bass.

2013

Résumé: The Nme gene family, also known as Nm23 or NDPK, is a very ancient gene family that can be found in all kingdoms of life. In the late eighties, a gene of the Nme family, NME1, was identified as the first metastatic suppressor gene, resulting in a major interest for this family. Due to the complexity of the family, the need for a unified and evolutionary-supported gene nomenclature was recently stressed by the scientific community. Based on a complete evolutionary history study of the gene family in metazoans and vertebrates, a unified nomenclature was recently proposed and accepted by gene nomenclature consortia. In addition to its well-documented role in tumor metastasis, members of the Nme family are also involved in a wide variety of cellular and physiological processes. Available data in non-mammalian species remain, however, scarce with the noticeable exception of Drosophila in which a major role in development was reported. In fish, very few studies have specifically investigated the role of nme genes. Several transcriptomic and proteomic studies have, however, revealed the expression of nme genes in various fish organs and tissues, in mature oocytes, and during embryonic development. Altogether, interest for the Nme gene family in fish is growing and new functions/roles in fish biology are expected to be discovered in the forthcoming years. Here, we briefly review the current knowledge of the Nme family in fish.

Résumé: The most commonly observed reproductive dysfunction in male fishes reared in captivity is reduction in sperm volume and quality. The Atlantic bluefin tuna Thunnus thynnus (Osteichthyes: Scombridae) is one of the few large pelagic and migratory marine fishes maintained in captivity with the purpose of establishing breeding populations to support an aquaculture industry. The objectives of the present study were to compare male germ cell proliferation and apoptosis between wild and captive individuals at two different phases of the spermatogenetic cycle, and to evaluate sperm motility characteristics of captive individuals. Histological observations were performed to analyze testicular activity, and germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferasemediated d'UTP nick end labeling (TUNEL) method, respectively. Computer-assisted sperm analysis (CASA) was used to evaluate sperm motility. Results showed that germ cell proliferation was delayed and germ cell apoptosis increased in captive animals relative to wild individuals. Sperm motility of samples obtained from captive individuals was anomalous, both in terms of motility duration and swimming efficiency. Thus it appears that rearing in captivity impairs male reproductive function through, at least, changes in germ cell proliferation and apoptosis.

2012

Résumé: The sperm of seabass is very fragile and it quickly loses its ability to fertilize after collection either if kept undiluted or in classic saline media. In order to avoid cryopreservation when only short conservation is required, the process of sperm management including sperm collection, sperm dilution rate in storage medium and storage medium composition, was subject to experimental trials. A concentration of 20% urine generated a low pH of seminal fluid, and it immediately altered the motility ability. However, pH did not seem to be the key agent of motility prevention since sperm dilution in Leibovitz culture medium (L15) or in classic saline medium both presenting a similar low pH (7.3) did not affect motility. L15 increased the duration of sperm survival by 2 days at 4 degrees C after collection. Moreover, dilution could be restricted to 1 : 3 (v : v) for conservation of chilled sperm. Chilled sperm could be cryopreserved with no more damages than those observed after freezing of fresh sperm.

Résumé: A controlled-release implant loaded with GnRH agonist (GnRHa) was used to induce spawning in Atlantic bluefin tuna (Thunnus thynnus) during two consecutive reproductive seasons. The fish were implanted underwater and sampled between days 2 and 8 after treatment. At the time of GnRHa treatment, females were in full vitellogenesis and males in spermiation. There was a rapid burst of pituitary luteinizing hormone (LH) release at day 2 after treatment in GnRHa-treated fish, and circulating LH remained elevated up to day 8 after treatment. In contrast, control fish had significantly lower levels in the plasma, but higher LH content in the pituitary, as observed in many other cultured fishes that fail to undergo oocyte maturation, ovulation and spawning unless induced by an exogenous GnRHa. Plasma testosterone (T) and 17 beta-estradiol (E(2)) were elevated in response to the GnRHa treatment in females, while 11-ketotestosterone (11-KT) but not T was elevated in males. Even though oocyte maturation and ovulation did occur in GnRHa-induced fish, no significant elevations in 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) or 17,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S), in either the free, conjugated or 5 beta-reduced,3 alpha-hydroxylated forms was observed in fish sampled within 6 days after treatment. Interestingly, a significant peak in plasma free 17,20 beta-P levels occurred in both males and females at day 8 after treatment. Histological sections of the ovaries in these females contained oocytes at the migrating germinal vesicle stage, suggesting the role of this hormone as a maturation-inducing steroid in Atlantic bluefin tuna. In conclusion, the GnRHa implants activated effectively the reproductive endocrine. axis in captive Atlantic bluefin tuna broodstocks, through stimulation of sustained elevations in plasma LH, which in turn evoked the synthesis and secretion of the relevant sex steroids leading to gamete maturation and release. (C) 2011 Elsevier Inc. All rights reserved.

Résumé: This work assesses the present knowledge on Pacific oyster sperm biology in comparison to two marine fish species (turbot and seabass) whose sperm characteristics are well described. Sperm morphology mainly differs by the presence of an acrosome in Pacific oyster which is absent in both fish species. In turbot as in Pacific oyster, a sperm maturation process along the genital tract is observed. Sperm motility is triggered by changes in osmolality for seabass and turbot and in pH for Pacific oyster. However, complementary factors are involved to maintain sperm immotile in the genital tract. Sperm movement duration is very long in Pacific oyster (2024 h), compared to turbot (35 min) and seabass (4050 s). A high capacity of ATP regeneration is observed in Pacific oyster sperm, sustained by the limited changes in its morphology observed at the end of the swimming phase. Then, the total distance covered by spermatozoa is very different among the studied species (seabass: 2 mm, turbot: 12 mm, Pacific oyster: 1 m). Considering the main characteristics of sperm movement, the three studied species can be separated in two groups: the sprint racer group (seabass: high velocity and short distance covered) and the marathonian racer one (Pacific oyster: low velocity but covering long distances). To an intermediate extent, turbot sperm belongs to the sprint racer group. Then, the two different sperm movement strategies observed in the three species, are compensated by the behaviour of the breeders.

2011

Résumé: After the recent report of the expression of several nme genes in the zebrafish gonads, the present study aimed at further analyzing the expression of nme genes in the ovary with special attention for the nme transcripts that are maternally inherited and could thus participate in the determination of oocyte developmental competence. The expression levels of all groups I and II nme genes were characterized by QPCR in a panel of zebrafish tissues. The nme genes exhibiting an ovarian expression were subsequently monitored throughout oogenesis and early development, and their expression sites characterized using in situ hybridization. Here, we show that nme2b1, nme3, nme4, and nme6 are highly expressed in the ovary and present in the zebrafish oocyte throughout oogenesis. While the four transcripts are maternally inherited, nme3 and nme6 display a typical maternal profile and are detected in the zebrafish early embryo. In contrast to nme3, nme6, abundance exhibits a sharp decrease during early embryogenesis. After zygotic genome activation, we observed an increased expression of nme2b1, nme2b2, nme3, and nme6. The present study provides a comprehensive overview of the expression of nme family members during zebrafish oogenesis and early development. In addition, the maternal origin of two nme transcripts in the early embryo is reported here for the first time in any vertebrate species. Together, our observations suggest an important role of the nme family in oocyte and embryo development in vertebrates.